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Zebra fish fatty acid desaturase gene, recombinant expression vector and application

A desaturase and expression vector technology, applied in the field of increasing the content of linoleic acid or α-linolenic acid in silkworm, desaturase gene and encoded protein, and can solve problems such as changing fatty acid content

Active Publication Date: 2015-10-21
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the expression of exogenous fatty acid desaturase gene in silkworm by silkworm transgenic technology, and then changing the fatty acid content through the expression of exogenous fatty acid desaturase gene

Method used

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  • Zebra fish fatty acid desaturase gene, recombinant expression vector and application
  • Zebra fish fatty acid desaturase gene, recombinant expression vector and application
  • Zebra fish fatty acid desaturase gene, recombinant expression vector and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Zebrafish fatty acid desaturase gene DrDes preparation.

[0033] Source gene: Zebrafish fatty acid desaturase gene sequence (NCBI accession number: NP_571720.2)

[0034] According to the codon preference of the coding gene expression sequence in the silkworm genome sequence data, the source gene was optimized and designed, and the zebrafish fatty acid desaturase gene DrDes was obtained after optimization. The nucleotide sequence is shown in SEQ ID No.1, named as DrDes gene.

[0035] The amino acid sequence of the protein encoded by the guide of DrDes gene is shown in SEQ ID No.2.

Embodiment 2

[0037] The silkworm transgenic recombinant vector pBac[3×p3-DsRed+IE1-DrDes-SV40] was constructed.

[0038] Such as figure 1 As shown, insert the DrDes gene into the multiple cloning site of the departure vector pSL1180 to obtain an intermediate vector, and then insert the complete expression cassette IE1-DrDes-SV40 containing the DrDes gene into the final vector pBac[3×P3-DsRed] to obtain recombination Expression vector.

[0039] Specific implementation operations:

[0040] Step S1, preparation of vector pSL1180[Ser1-DrDes-SV40]

[0041] The DrDes gene was connected to the pUC57 vector plasmid; the pUC57 vector plasmid was digested by BamHI / NotI double enzymes, and the DrDes gene fragment was recovered (the recovery operation was performed according to the instructions of the TAKARA gel recovery (small amount) kit), and the recovered fragment was digested with the same enzyme The pSL1180[Ser1-pGH-SV40] vector backbone fragment was ligated according to the instructions of T...

Embodiment 3

[0059] The silkworm transgenic recombinant vector pBac[3×p3-EGFP+Lp3-DrDes-SV40] was constructed.

[0060] Such as figure 2 As shown, insert the DrDes gene into the multiple cloning site of the departure vector pSL1180 to obtain an intermediate vector, or insert the complete expression cassette Lp3-DrDes-SV40 containing the DrDes gene into the final vector pBac[3×P3-EGFP] to obtain Recombinant expression vector.

[0061] Specific implementation operations:

[0062] Step S1, preparation of vector pSL1180[Ser1-DrDes-SV40]

[0063] Same as embodiment two.

[0064] Step S2, preparing the Lp3 promoter sequence

[0065] PCR amplification in vitro, the Lp3 promoter sequence was amplified from the BmNPV genome, and EcoRI and BamHI restriction sites were added to the upstream and downstream of the amplified fragment respectively, and the plasmid was obtained by T-A cloning into the vector pMD19-T, and the obtained pMD19-T The vector plasmid was digested with EcoRI and BamHI; the ...

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Abstract

The invention discloses a zebra fish fatty acid desaturase gene which is named a DrDes gene with the nucleotide sequence of SEQ ID No.1. The DrDes gene is the new desaturase gene which is obtained by optimizing and designing a fatty acid desaturase gene sequence of zebra fish according to the preference of encoding gene expression sequence codons in silkworm genomic sequence data. The nucleotide sequence of proteins guided by the DrDes gene to be encoded is SEQ ID No.2. The invention meanwhile provides a recombinant expression vector with the DrDes gene and an establishing method of the recombinant expression vector. The promoter of an expression frame of the recombinant expression vector is a BmNPV very-early promoter IE1 or a silkworm fatty body tissue specificity promoter Lp3. The recombinant expression vector with the DrDes gene can be used for obtaining a transgenic silkworm variety with high linoleic acid content or high alpha-linolenic acid content. The invention further provides a method for increasing the linoleic acid content or the alpha-linolenic acid content in silkworm tissue. The target transgenic silkworm variety is obtained through screening by introducing the zebra fish fatty acid desaturase DrDes gene to silkworm embryo.

Description

technical field [0001] The invention relates to a desaturase gene and encoded protein, and a method for increasing the content of silkworm linoleic acid or alpha-linolenic acid realized by the same. It belongs to the field of genetic engineering and transgenic animal breeding. Background technique [0002] In organisms, many metabolic processes are carried out on or related to membranes, and unsaturated fatty acids are important components of cell membrane phospholipids. Since the freezing point of fatty acids decreases with increasing unsaturation, the higher the content of unsaturated fatty acids in membrane lipids, the lower its phase transition temperature and the greater its fluidity. Furthermore, various functions of the cell membrane, such as cell recognition, translocation, and activity of membrane-bound enzymes, are closely related to the fluidity of the cell membrane, so unsaturated fatty acids play important functions directly or indirectly in organisms, such as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/85C12N15/66A01K67/04
Inventor 黄先智于新波沈以红
Owner SOUTHWEST UNIVERSITY
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